In my neutrophil ROS production assay, fluorescence of the no cell control with DHR is higher than the cell stained with DHR? Basically, column 2 is always higher than column 4.
Example:
Column 1: no cell
Column 2: no cell (replaced with media) + DHR
Column 3: cell + DHR + PMA
Column 4: cell + DHR
The cell treated with PMA and stained with DHR read more than 100 fold than the controls, so that doesn't seem to be a problem. I have performed this 12 times, and 2 of the 12 attempts column 2 had lower reading.
Why is this happening?
Thing I have ruled out:
1. Sample
2. protocol of the reader
3. All fresh reagents
4. staining or plating protocol. Other have used the same protocol in the lab.
Thank you for your help in advance. I welcome any suggestions.
Consulted with an expert in the field. Fluorescence can get trapped in the membrane which explains the lower fluorescence detected in the cell+dhr control than nocell+dhr control.
Cortisol concentration in the animals may have an affect on the membrane trapping the fluorescence molecules.
This is not an uncommon issue in this assay.
Hey Theros,
ROS measurements are done routinely in our lab (with macrophages however) but it took a long time to set it right.
We used Dihydrorhodamine once (not fixaded though) to measure ROS, but were not satisfied with it. First of all it diffuses everywhere (in the cell, outside the cell, over every membrane in the cell), so you can just measure "the overall" ROS in the cell. In our hands this was not useful, because in macrophages and neutrophils different ROS sources are activated in different compartments.
We sucessfully measured ROS with macrophages, neutrophils and human PBMCs with our commonly used ROS probes (Isoluminol for extracellular ROS, 6,5-Dicarboxy-DCF for cystosolic ROS and MitoSOX for MitoMatrix ROS) with bacterial infection (or PMA as a chemical stimulus) and this worked quite well. There are other probes like DHE, wich I would not tecommend. The problem with luminol is, that it freely diffuses over cell membranes. So by using this substance you get a "overall ROS measurement". The same goes with the generally used DCF substance H2DCFDA, which a lot of people use. This also freely diffuses over membranes, so with that DCF probe you only detect "overall ROS" in the cell. For both ROS probes derivates exist that are not cell permeable (isoluminol) or are retained only in the cytosol (6-Carboxy-DCF).
Another concern about ROS level determinations either with Microscopy or FACS is, that they are only snapshots of a rapid response. Macrophages, neutrophils and PBMCs react very quick (at least after infection and PMA) with ROS production (peak after 30 min) and end approximately after one and a half our. I would always recommend ROS measurements in a plate reader with kinetics (it worked with PBMCs and neutrophils in our hands) instead of making "ROS-Snapshots".
For ROS measuremtents in a plate reader do not use serum-containing medium. We use HBSS and measure ROS for the maximum of 2 hours.
For any information or techniqual detail for ROS measurements in a plate reader (with different stimuli, scavengers and in different compartments), I would like to highly recommend our paper. 80 % of it is more or less about ROS measurements in a plate reader. The protocols described there worked in our hands with macrophages, neutrophils, human PBMCs, Microglia and MEFs.
https://stke.sciencemag.org/content/12/568/eaar5926
Mitochondrial reactive oxygen species enable proinflammatory signaling through disulfide linkage of NEMO.
Please keep me informed, how it goes and ask any time for further help.
All the best,
Marc
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